Figure 6.
Mutation of the E2F-2 Element Alters the Developmental Regulation and TGMV Activation of the N. benthamiana PCNA Promoter.
(A) Electrophoretic mobility shift assays with 32P-labeled E2F-2 DNA alone (lane 1) or incubated with N. benthamiana nuclear protein extract (lanes 2 to 11). Lane 2 had no added competitor DNA. Lanes 3 to 5 contained unlabeled E2F-2 DNA (wt). Lanes 6 to 8 contained unlabeled mutant E2F-2 DNA (mt). Lanes 9 to 11 contained a random control DNA (c). Competitor DNAs were used at 10-, 100-, or 250-fold molar excess relative to probe, as indicated by the triangles at the top of the gel. The shifted product corresponding to the specific E2F-2/protein complex is marked by the arrow.
(B) The ratios of Luc activities in young versus mature leaves of N. benthamiana lines (left) carrying the mutant E2F-2 −633 PCNA:luc construct are shown. The dotted line (mt) corresponds to the mean ratio for plants transformed with the mutant construct, whereas the solid line (wt) correlates with the mean ratio for plants transformed with the wild-type −633 PCNA:luc construct (Figure 3B). The error bars indicate two standard errors.
(C) Soluble protein extracts from N. benthamiana lines carrying the mutant E2F-2 −633 PCNA:luc construct were assayed for Luc activity and compared with lines carrying the wild-type −633 PCNA:luc construct shown in Figure 3B. The Luc-specific activity values from young (y) and mature (m) leaves of the mutant (mt) and wild-type (wt) constructs are compared in the scattergram. The eight independent lines given in (B) are shown for each treatment. The symbols indicate different lines.
(D) P values of t test comparisons of the data shown in (C). Statistically significant values (P < 0.05) are shown in boldface type.
(E) Developmental regulation of the mutant E2F-2 PCNA promoter in untreated, mock-inoculated, and infected plants (14 days after inoculation). Soluble protein extracts from individual leaves of untreated (crosses), mock-inoculated (open circles), and infected (closed circles) mt-16 (left) and mt-35 (right) plants carrying the mutant E2F-2 −633 PCNA:luc transgene were assayed for Luc activity. Each point represents the average of two plants. (Note the different scales relative to Figure 5C.) A gradual decrease in PCNA activity with leaf number and little effect of TGMV infection also were seen for line mt-29 (our unpublished data).